The present invention relates to a confocal microscope apparatus and a photographing apparatus for a confocal microscope, for photographing a confocal image of a microscope specimen.
In recent years, confocal microscopes have rapidly spread owing to their improved effects in optical performance called confocal effects including a sectioning effect along the optical axis and a super-resolution effect. These confocal microscopes can be roughly classified into two, single-beam type (fixed-pinhole type/a single pinhole) and multi-beam type (movable pinhole type/a plurality of pinholes) on the basis of the scanning principle and the basic arrangements of a scanner and an optical system.
The single-beam type confocal microscope drives a laser beam for scanning, using a galvanometer mirror or an optical deflector such as an acousto-optical deflector (AOD). The-multi-beam type confocal microscope uses a rotating disk scanner (to be referred to as a “disk scanner”) represented by a scanner which rotates a Nipkow's disk on which a plurality of pinholes are formed in a spiral pattern.
The latter confocal microscope using the rotating disk scanner, and particularly a multi-beam type confocal microscope using a disk scanner with a microlens is described in detail in “Yokogawa's Confocal Microscope” (Japan Industrial Publishing, “Optical Alliance”, Vol. 7, No. 12), and “Nipkow's Disk Type Confocal Fluorescence Microscope” (Japan Industrial Publishing, “Optical Alliance”, Vol. 8, No. 10). A most significant feature of this type is that this microscope enables direct observation with the naked eye and photography, and enables observation and photography of a color image.
For this reason, the multi-beam type confocal microscope with a disk scanner whose main object is visual inspection is popular for industrial purposes, and particularly semiconductor inspection purposes of ICs and the like. To the contrary, the single-beam type confocal microscope is still popular in medical and biological study purposes which have strong demands for observing, e.g., a cell dyed with a fluorochrome at high image quality to photograph a still image.
Recently, as described in the above references, a multi-beam type confocal microscope using a disk scanner with a microlens, suitable for fluorescent observation, has appeared and is also used in medical and biological study purposes. However, the application is often limited to real-time observation at a video rate or more for the movement of a cell injected with a fluorescent indicator such as Ca ions or pH.
This arises from marketing factors such as a small number of manufacturers for confocal microscopes using disk scanners, resulting in few product variations, and poor balance between apparatus cost and performance, resulting in low cost performance. However, it should also be noted that the disk scanner design currently assumes operation in combination with a real-time moving picture photographing apparatus such as a video camera.
For example, in the technique disclosed in Jpn. Pat. Appln. KOKAI Publication No. 9-80315, as shown in FIG. 6, rotation of a Nipknow's disk 120 is detected by a photodiode 121 to generate a trigger signal to an image sensing device 125 using a current-to-voltage converter 122 and a voltage comparator 123. The scan period and image sensing period are synchronized to eliminate any scan fluctuation and obtain a sensed image free from any bright/dark stripe.
The image sensing device 125 assumed in this technique continuously photographs at a predetermined period, i.e., 1/30 sec, like a video camera using a CCD. That is, this technique does not consider use conditions for photographing a still image, and particularly photographing conditions under which the exposure time changes over a wide range of several hundredth sec to several ten sec in accordance with the specimen brightness.
In the technique disclosed in Jpn. Pat. Appln. KOKAI Publication No. 9-297267, as shown in FIG. 7, a vertical sync signal is extracted (202) from a video signal (NTSC signal) from a CCD camera 201 of a video photographing device, and the frequency of the extracted signal is multiplied (integer multiple) (203) to supply a control signal to a motor driver 204 for driving a disk scanner. The number of turns of a motor 205 can be controlled based on the NTSC signal to realize a motor control device capable of obtaining an image free from any scanner fluctuation and stripe.
Again, however, the image sensing device is limited to the CCD camera 201 using a NTSC signal, and does not consider any application purpose of photographing a still image whose exposure time changes over a wide range.
As described above, since the conventional disk scanner assumes the CCD camera 201 as an image sensing device, the rotational speed and rotational period of the scanner are set to about 30 rps corresponding to a video rate (about 30 Hz), and about 1/30 sec (about 0.033 sec), respectively. Fine adjustment of the rotational speed in synchronism with the CCD camera 201 is possible, but a change of the rotational speed over a wide range is not possible.
An explanation will be given to a problem arising when the exposure time changes over a wide range of several hundredth sec to several ten sec depending on the specimen brightness, similar to the use conditions of general micrography.
Assume that the disk scanner rotates at about 30 rps corresponding to a video rate (about 30 Hz). For an exposure time of about 0.01 sec (1/100 sec), the scanner rotates only about 0.3 times within the exposure time. For an exposure time of 10 sec, the scanner rotates about 300 times within the exposure time. For this reason, when the exposure time changes over a wide range, the number of turns of the scanner while obtaining an image greatly changes depending on the exposure time.
Image noise such as stripes caused by a nonuniform layout of pinholes on the disk scanner in the prior arts of the above references may be accumulated and eliminated by increasing the number of scan operations for a scan pattern (scan track) on the scanner, i.e., increasing the total number of turns of the scanner within the exposure time for photographing one image. For this reason, when the specimen is bright and the exposure time is short, the noise accumulation and elimination effect degrades as the total number of turns of the disk within the exposure time decreases. As a result, image noise by the stripes of the pinhole pattern stands out, and a high-quality still image is difficult to obtain.
Especially when the exposure time is only 0.01 sec (1/100 sec), the scanner rotates only about 0.3 times within the exposure time, and the number of turns of the disk is a decimal fraction smaller than 1. In this case, even if the exposure start timing is aligned with the start point of a scan track, the exposure end timing does not coincide with the end point of the scan track.
Assume that the start and end points of scan tracks are set at a plurality of portions, e.g., at intervals of a rotation angle of 30° (12 scan tracks per turn) for one disk turn, and the end point of a scan track accidentally coincide with the exposure end timing for an exposure time of 0.0833 sec (1/12 sec). Even in this case, if the specimen brightness slightly changes to change the exposure time, the end point of the scan track immediately shifts from the exposure end timing.
More specifically, when the exposure time changes depending on the specimen brightness in still image photography by a conventional multi-beam type confocal microscope using a disk scanner, the start and end points of the scan track cannot always coincide with the exposure start and end timings. Particularly for a short exposure time, simultaneously when the number of scan track scanning operations decreases, the exposure start and end timings shift from the start and end points of the scan track. Then, image noise such as stripes caused by a nonuniform pinhole layout is easily superposed on an image, failing to obtain a high-quality still image.